Performance-Verified High-Speed LC: Assessing the Capabilities of a Flexible System for STM-LC and HPLC

Analytical laboratories are increasingly challenged to improve data quality and increase sample throughput. Exploration of new ways to meet these requirements has generated considerable interest in liquid chromatography systems utilizing sub-two-micron (STM/<2 μm) column technology. Providers of these instruments claim reduced run times, improved resolution, as well as other performance enhancements.

Because questions have been raised concerning the accuracy and specificity of some of these claims, there has been a need for objective criteria by which to gauge performance of the various STM-capable LC instruments. In the article in Ref. 1, authors from a large pharmaceutical company outline what they consider to be levels of performance indicative of the potential of STM technology. Their criteria include:

  • Demonstrating instrumental capability for generating high LC flow rates for faster separations while minimizing increases in backpressure
  • Utilizing elevated temperature to reduce separation times
  • Accommodating all HPLC and STM-LC operational modes on a
    single instrument and facilitating use of HPLC methods without the need for revalidation
  • Achieving separation efficiencies close to theoretical predictions
  • Demonstrating the high-speed STM capability without compromising precision or sensitivity.

To these can be added:

  • Maximizing sample throughput by minimizing cycle times
  • Ensuring ease of use
  • Providing robust system and data security
  • Minimizing downtime through proactive diagnostic monitoring and maintenance.

Defining terms

Rapid Resolution liquid chromatography (RRLC, Agilent Technologies, Palo Alto, CA), also known as ultra high pressure chromatography (U-HPLC) or ultrafast LC (UFLC), are the terms used to designate LC applications employing STM columns with 1.0–4.6 mm i.d.

Pump pressure

LC run times are primarily a function of flow rate. With 3.5-μm or 5.0-μm column packing, increases in linear velocity beyond approximately 1.0 mL/min are accompanied by decreases in column efficiency, which negatively affects resolution. At flow rates of about 2.0 mL/min, the decrease in column efficiency for 3.5-μm and 5.0-μm particles is roughly 15% and 40%, respectively. At a flow rate of 5.0 mL/min, the corresponding losses are 40% and 65%, respectively. This is not the case for STM columns, for which the efficiency remains fairly constant over the same flow rate range (Figure 1). As a result, STM columns make it possible to shorten run times considerably without compromising resolution. (A 5–10 fold run time decrease is typical, even without increasing temperature.) Sustaining faster linear flow requires adequate pumping capacity.

Figure 1 - Column efficiency at increasing flow rate for conventional HPLC vs STM-LC columns. Unlike columns with 5-μm or 3.5-μm packing, STM-packed columns are efficient at high linear flows and realize significant gains in analysis speed, typically 5–10× without changing temperature. The STM column used here is a Zorbax 1.8-μm RRHT (Agilent Technologies) with 4.6 mm i.d. Experiments were performed on an Agilent 1100 series binary LC system.

At a given pumping pressure, a decrease in packing particle size will cause a corresponding drop in flow rate. To overcome this effect, additional increases in pumping pressure are needed. Moreover, as linear speeds increase, more stringent control of flow rate variations and other deviations in chromatographic dynamics are necessary to maintain acceptable precision. These conditions can be met by a binary pump capable of producing pressures up to 600 bar and flow rates up to 5 mL/min. Perturbations to constant flow are held within narrow limits by the pump’s electronic dampening control system. The result is precisely reproducible chromatographic performance (Figure 2) as well as lower baseline noise.

Figure 2 - Maintaining precision despite significant changes in flow rate and temperature. The 1200 series RRLC is equipped with electronic dampening of the system pump piston to minimize flow rate fluctuations and electronic controls that hold temperature constant to within ±0.05 °C. These system constraints also lower pump and heater contributions to baseline noise.

While brute pumping force is required to generate the pressures needed for faster chromatographic flow rates, improvements in pumping efficiency need to be considered as well. Optimizing the instrument configuration to minimize its contribution to the overall backpressure enables greater utilization of the pump’s capacity for generating faster flows. Reducing the load on the pump also contributes to longer system and component life and lowers both maintenance downtime and operating costs. To that end, an RRLC system incorporates additional pressure reducing characteristics such as:

  • More efficient packing of the column and the use of STM columns with particle size distributions designed for pressure reduction (Zorbax 1.8-μm RRHT columns)
  • Utilizing system-connecting capillaries with internal diameters and lengths that produce lower backpressure without increasing peak dispersion
  • Autosampler valving and electronic controls designed for minimal injection pressure spikes.

The combination of higher pumping capacity and system-engineered pressure lowering enables an RRLC system to operate efficiently over a flow rate range of 0.05–5 mL/min, giving linear flow rates of up to 16 mm/sec at absolute flow rates of up to 2.4 mL/min (5 mL/min) on 2.1-mm (3-mm) i.d. RRHT columns.

Temperature

Because of its inverse relationship to mobile phase viscosity, temperature is another parameter that can be manipulated to increase flow rate. (This may not apply to non-Newtonian fluids such as polymers and substances that form immobilized bridged networks such as gels.) This is especially the case for important chromatographic solvents such as water and alcohols that exhibit strong intermolecularly bonded networks that are considerably weakened, with commensurate reduction in viscosity as the temperature is increased.

The equation t/N ∝ η, which describes the relationship between temperature and viscosity, merits further attention (t = temperature, N = theoretical plate number, and η is viscosity). Rearranging the terms t/ηN yields an expression indicating that column efficiency increases rapidly with increasing temperature since an increase in t is accompanied by a decrease in η. Figure 3 illustrates the run time reductions gained by the use of an STM packing in a narrow-bore/short-column format and the additional reductions realized by running this setup at elevated temperature. Note that the decreases in run times are achieved with resolution comparable to HPLC. In certain applications, such as LC-electrospray ionization (ESI)-MS, elevating the temperature can also improve sensitivity by increasing the efficiency of nebulization and desolvation in the electrospray ionization process.

Figure 3 - Maximizing run time reduction. Performing STM-LC in a short, narrow-bore column at modestly elevated temperature achieves a tenfold decrease in run times on the 1200 series RRLC. Using the same chromatographic configuration and elevating the temperature results in a 27-fold increase in analytical speed compared with HPLC performed on a standard-bore column with a standard particle size packing. STM-LC/temperature gains in analytical speed do not come at the expense of resolution, sensitivity, or precision, which are comparable to the corresponding HPLC method.

High-temperature LC introduces a set of constraints that must be properly managed to avoid potentially counterproductive effects. First and foremost, the column packing chemistry must be thermally stable. Performance specifications for Zorbax Stablebond C18 columns used in the experiments presented here demonstrate long-term stable operation at temperatures up to 90 °C and beyond. The LC column temperature can be rapidly elevated by means of a thermostated column compartment equipped with Peltier-heating technology. A low-volume heat exchanger is employed to minimize peak broadening on narrow-bore columns.

A second Peltier-controlled heat exchanger incorporated postcolumn forms a feedback loop with the flow cell temperature sensor, ensuring optimal thermostating of the eluent and the elimination of temperature-induced noise in the detector. Low-noise electronics complement the flow cells that have been redesigned to maximize sensitivity. Postcolumn cooling coupled with fast run times also significantly reduce the probability of degradation by limiting analyte residence time at elevated temperature.

Accommodating conventional HPLC and RRLC operational modes

Instrument flexibility is an especially important attribute in a period of significant technology transition. The ability to accept both narrow- and standard-bore columns eliminates the need for separate HPLC and RRLC instruments. Users can continue to run current HPLC methods, maintain the existing work flow, and meet regulatory requirements while simultaneously making the transition to RRLC.

Figure 4 - Selectable delay volume avoids retention time mismatch when migrating methods from HPLC to RRLC. RT shifts of up to 25% can occur when (a) an inappropriately low delay volume is used for running RRLC on standard-bore columns. The RT discrepancy limits compatibility between HPLC and RRLC applications and confounds peak tracking, requiring method revalidation. Selection of the appropriate 600–800 μL standard delay volume (b) for both RRLC and conventional HPLC methods run on standard-bore columns aligns run times, eliminating the need for method revalidation. The lower 120-μL delay volume is reserved for narrow-bore column LC and LC-MS applications.

Adapting standard-bore column methods to narrow-bore columns ordinarily results in significant alterations in retention time. (Shifts as large as 30% have been documented.) This large displacement creates considerable uncertainty with respect to peak identification, necessitating method revalidation (Figure 4a). One way to overcome this problem is to employ selectable delay volumes: 600–800 μL for standard-bore columns and 120 μL for narrow-bore columns. Using the standard delay volume eliminates the HPLC-to-RRLC retention time displacement, which is typically held to <2%. As a result, existing HPLC methods can be executed without revalidation, and a method changeover from HPLC to RRLC often requires as little as 5 min (Figure 4b).

Figure 5 - Near theoretical separation efficiencies for STM-LC in both a) narrow-bore and b) standard-bore columns. 1200 RRLC performance in standard-bore columns shows no sacrifice in chromatographic efficiency as a result of a thermal mismatch between mobile phase and column. (Such a thermal mismatch would cause band broadening due to temperature gradients across the column radius.) The data shown here demonstrate nearly comparable column efficiency and resolution, approaching theoretical values for both standard- and narrow-bore STM columns. Apparatus/experimental conditions—Test sample: set of nine compounds, 100 ng/μL each, dissolved in acetonitrile (ACN). 1) Acetanilide, 2) acetophenone, 3) propiophenone, 4) butyrophenone, 5) benzophenone, 6) valerophenone, 7) hexanophenone, 8) heptanophenone, 9) octanophenone. Instrument: 1200 Series RRLC with binary SL pump, high-performance Autosampler SL, thermostated column compartment, diode array detector SL (13-μL cell, 20-Hz data acquisition rate slit: 4 nm, signal: 245 nm). Columns: 4.6 and 2.1 × 150 mm SB-C18, 5 μm and 1.8 μm. Solvent A: H2O + 0.1% trifluoracetic acid (TFA). Solvent B: ACN + 0.095% TFA. Gradient: 50–95% B in 7 min, hold over 1 min. Stop time: 10 min. Flow rate: 1.5 mL/min. Injection volume: 3 μL. Wash: 5 sec. Temperature: 50 °C.

Achieving separation efficiencies close to theoretical prediction

Run time reduction is not the only objective of STM technology. The term “rapid resolution” indicates that improved separation efficiency is also an important objective. Figure 5a and b compare runs on 5-μm and 1.8-μm Zorbax 2.1 × 150 mm and 4.6 × 150 SB-C18 columns, demonstrating efficiency gains close to theoretical prediction on both narrow- and standard-bore columns. Figure 5 also demonstrates that the highest absolute resolution is achieved on 4.6-mm columns, which show an inherently higher efficiency than 2.1-mm columns.

Figure 6 - Accurate impurity quantitation with rapid run times. Under ultrafast LC conditions, the 1200 Series RRLC equipped with an Agilent DAD/MWD SL allows accurate quantitation of impurities and side products at levels smaller than 0.05% of the main compound(s).

As retention times decrease, peaks narrow (0.2–1 sec widths are typical) and detection sampling rates must be increased to preserve resolution. Use of a low-noise diode array detector (DAD) that can sample up to 80 Hz makes it possible to routinely achieve a S/N >10 at 0.5 mAU or 0.03% level, without dispersing peaks by inappropriate low sampling rates. This is in excess of the sensitivity required for quantification of pharmaceutical side products below the critical 0.05% of the main compound (Figure 6). Table 1 provides further insight into the tradeoff between data sampling rate, data quality, and sensitivity. It shows that a properly designed LC system can provide significant flexibility to adjust these parameters for various application requirements.

Fast run times: No assurance of high analytical throughput

Minimizing turnaround time is vital if the savings from fast run times are to be translated into high-volume sample throughput. One approach to reducing cycle times for high-throughput operations is called alternating column regeneration (ACR). This is a tandem, dual-column configuration in which one column is flushed and equilibrated while the other performs a chromatographic separation. ACR can reduce cycle times by as much as 50% and produce throughputs of more than 2000 samples per day.

ACR is enabled by means of a two-position/10-port valve system that controls the timing and fluid paths required for each phase of the chromatography and/or column regeneration (Figure 7). Because column flush and regeneration intervals are typically shorter than analysis times, additional means may be employed to further shrink cycle times. Examples include using an automatic delay volume reduction that bypasses the sampling assembly directly after the sample has been injected and before the gradient is introduced onto the column (6 μL on the 1200 highperformance Autosampler SL) and the ability to prepare the next injection while the analysis takes place in bypass mode, which is typically referred to as overlapped injections.

Figure 7 - ACR-enabling two-position/10-port valve system. ACR saves up to 50% of cycle time by utilizing parallel column wash and regeneration, overlapped injections, and accelerated sampling operations. Throughputs of more than 2000 samples/day can be achieved.

Reducing the intervals for operations such as sampling and column regeneration requires additional measures to ensure that injections remain precise and that carryover between successive runs is minimized. In certain instances, both objectives can be facilitated with the same technology solution. For example, directing solvent flow through the sampling assembly (needle, loop, and metering device) ensures continual flushing of the internal surfaces. Simultaneous external washing of the needle helps prevent contamination of the needle seat. Automatic additional valve switches at different programmable or automatic time points provide the means for further flushing persistent residues from the system. High-throughput operations are also beneficially supported by the use of large solvent reservoirs that permit unattended operation for extended periods.

Control, compliance, usability, and servicing issues

System design that incorporates robust user support helps to maximize performance and ease of use while minimizing downtime and operating costs. Especially useful are instrument controller enhancements such as highly readable displays and the elimination of time-wasting routines such as those requiring system restarts whenever a configuration is changed. Advances in electronics make a faster software response possible, and the extension of centralized control to a larger universe of instrument components. Controllers that incorporate these innovations enable more rapid and comprehensive data management (analysis, retrieval, and review) and improved system navigation.

LC instruments at the cutting edge now offer a multiplicity of interactive help functions that gauge performance; proactively indicate when maintenance is needed; and provide straightforward, user-guided diagnostics. These routines increase the efficiency with which the instrument asset is managed by implementing timely parts replacement and rapid deployment of the requisite servicing resource, when necessary.

A number of measures are increasingly seen as intrinsic to secure and compliant instrument and laboratory operations. These include data loss protection in the event of a communication interrupt and electronic tags that unambiguously track system parameters. Data and system security are addressed through multiple layers of protection, including Internet firewalls, user control of communications, end-to-end data encryption, and compliance with network security. Access is typically password protected and can be ranked according to a system of privileges.

Conclusion

The data presented here demonstrate unequivocally that the 1200 series RRLC meets the range of criteria for delivering the full performance potential of STM technology as well as supporting conventional HPLC. It also shows that the system provides an easy and secure transition between the two technologies. This flexibility derives from the instrument platform design that comprises a matrix of functional modules that can be configured and reconfigured to meet a diversity of analytical LC applications. The various configurations constitute a continuum or glide path across performance regimes that address current and anticipate future needs. Users entering the 1200 series continuum with an 1100 series emulation or by upgrading an 1100 series instrument with 1200 series modules are well positioned to reconfigure their system for RRLC operations. Naturally, one can start out with a fully equipped 1200 RRLC system configuration.

Reference

  1. Kofman, J.; Zhao, Y.; Maloney, T.; Baumgartner, T.; Bujalski, R. Ultra-high performance liquid chromatography: hope or hype? http://www.chem.agilent.com/Library/articlereprints/Public/5989-5169EN-lo.pdf.

Dr. Frank is Application Chemist, Dr. Gratzfeld-Huesgen is Application Chemist, and Dr. Schuette is Program Manager 1200 Series Rapid Resolution System, Agilent Technologies, Hewlett-Packard-Str. 8, 76337 Waldbronn, Germany; tel.: +49 7243 602820, ext. 510; e-mail: [email protected].